Prokaryotic genome expression

Question 1

RNA polymerases is made up of which subunits?

Answer 1

alpha2BB’gammasigma

Question 2

Which sigma factor recognises -24 and -12 sites

Answer 2

SIgma 54

Question 3

How does Spo1 ensure its genes are expressed in the correct order

Answer 3

Uses alternative sigma factors encoded within the genes previously transcribed.

Sigma70 is used to binds the promoter of the early genes. Early genes expressed and Sigma middle produced, which can bind core polymerase and compete with sigma 70. Holoenzyme with sigma middle expresses the middle genes. Middle genes contain a sigma late, which can bind with core polymerase and transcribe the late genes.

Question 4

How does lamba phage ensure its genes are expressed in the correct order?

Answer 4

Uses an antitermination protein, N-protein. N protein binds to mRNA release channel and elomngates it so that the intrinsic stem hairpin loop folds after the UUUU sequence has been transcribed and a heteroduplex with stronger H bonding is being transcirbed. Interacts with a Nut Box (N-utilisation)

Question 5

Name 4 ways in which transcription can be terminated. Describe briefly…

Answer 5

Rho independent- intrinsic termination with stem hairpin loop and UUU sequence.
Rho dependent- Rho atpase binds Rut site and chases RNAP. Hairpin without the U’s acts as a pause site to halt RNAP and Rho collides with it and releases the mRNA

Riboswitches (attenuation)- Example SAM and methionine biosynthetic apparatus expression

Attenuation by ribosomes. Leader peptide with Trp. If ribosome produces leader it remains behind RNAP and prevents 2:3 hairpin, 3:4 hairpin acts as a terminator as enough Trp present. If leader isnt made, Ribosome stays at region 1 and 2:3 hairpin forms and RNAP can transcribe the Trp operon.

Question 6

Dsecribe the structure of the lac repressor

Answer 6

Tetramer (dimer of dimers).
Head peiece interacts with the major groove via a HTH motif
Hinge helix- 2 come together in the dimer and sit in the minor groove, distorting the DNA
N subdomain
C subdomain- Inducer binds between the 2 subdomains
Tetramerisation domain- helix enabling 4 monomers to assemble together

Question 7

Distance from O1 to O2 lac operators

Answer 7

400bp downstream

Question 8

Distance from O1 to O3 lac operators

Answer 8

90bp upstream

Question 9

-10 region core promoter sequence

Answer 9

TATAAT

Question 10

-35 region sequence

Answer 10

TTGACA

Question 11

Changes in Kd due to

Answer 11

Changes in the sequence of Up elements or -35 region

Question 12

Changes in k2 due to

Answer 12

Changes in the sequence of the -10 region

Question 13

Repressors influence k2/kd/both

Answer 13

Kd only, prevent RNAP binding

Question 14

Activators influence k2/kd/both

Answer 14

Both, increase affinity of RNAP for promoter and incerase rate of isomerisation

Question 15

RNAP footprint

Answer 15

-50 to +20

Question 16

Lac repressor footprint

Answer 16

-3 to +24

Question 17

Trp repressors co repressor

Answer 17

Trp

Question 18

CAP binds DNA via

Answer 18

Helix turn helix motif

Question 19

Describe the action of MerR

Answer 19

MerR binds DNA always, on opposite side of strand to RNAP binding. In MerR form it only binds and -10 to -35 region is 19bp. If mercury binds to MerR it twists and distorts the DNA to produce a difference of 17bp and promoting the binding of RNAP

Question 20

How do you regulate the regulators?

Answer 20

Bind small molecules e.g. SAM, mer, cAMP
Covalently modify them, e.g. activate by phosphorylation
Control the concentration of the protein by degradation or controlling the initial synthesis

Question 21

Trp repressor binds which promoters

Answer 21

Promoter for Trp operon, TrpR and aromamtic synthesis

Question 22

Describe activators that bind to sigma54

Answer 22

ATP utilizing, distance and have to bend DNA to bring them in close proximity. Finger like projections that hydrolyse ATP, similar to eukaryotic enhancers.